Fructans are polymers of fructose which are synthesized from sucrose and used as storage or reserve carbohydrates by many plants. They consist of fructosyl residues polymerized to sucrose, and therefore comprise fructosyl units in addition to one glucose unit. In view of this composition, they are highly soluble in water. The linkages between the fructosyl-residues are either exclusively of the β(1-2) type forming a linear molecule (inulin) in which the fructosyl residues are attached to the fructosyl residue of the sucrose starter, or of the β(2-6) type (levan), or both linkage types occur in branched fructans (graminans). Inulins are present in plants belonging to the Asterales (e.g. chicory) or the Liliaceae (e.g. onion). All fructans found in the dicotyledons, as well as some monocotyledons are of this type. The inulin in onion is termed neo-series inulin and has two linear β(1-2)-linked fructosyl chains, one attached to the C1 of the fructosyl residue of the sucrose and one attached to the C6 of the glucosyl residue of the sucrose. Levans are typically found in monocotyledons such as the Poaceae (e.g. grasses) and in almost all bacterial fructans. Graminans which consist of β(2-6)-linked fructose units with β(1-2) branches and are therefore more complex structures can also be present in cereals, and can be mixed with levans.
The degree of polymerization (DP) and distribution of linkage types are characteristic of different plant species. Since a range of DP are often seen in any one species, fructans typically show a disperse molecular weight. In contrast to the high molecular weight of fructan (levan, 1-5×106 Da) elaborated as an extracellular polysaccharide by some bacteria, plant fructans are much smaller by 2-3 orders of magnitude.
Fructans, rather than starch, occur naturally as the primary reserve carbohydrate in about 10-15% of higher plants including chicory, artichoke, asparagus, dahlia and the onion family, primarily in the perennating organs. Fructans are mostly stored in taproots (e.g. chicory) or tubers (e.g. dahlia, Jerusalem artichoke) or bulbs (e.g. onion). In grasses and cereals, fructans are mainly stored in the stems and leaf bases and used as a reserve carbohydrate for growth and seed production. Fructan also occurs as a temporary storage form in the vegetative tissues of forage grasses and cereals, but only at low levels in cereal grain. Despite this, wheat products are the primary source of fructan in the Western diet. Onions are the second largest source of naturally occurring fructans in the American diet, accounting for about 25% of total consumption (Moshfegh et al., J Nutr. 129(Suppl): 1407S-11S, 1999).
Cereals such as wheat and barley accumulate, mainly in vegetative tissues, branched graminan-type fructans containing both β-(2,1) and β-(2,6) fructosyl linkages. These mostly have a low DP, such as 1-6-kestotetraose (bifurcose) which is the major fructan oligosaccharide accumulating in crown tissues and leaves of cereals exposed to chilling. Fructans are naturally present in various cereal grains (White and Secor, Arch Biochem Biophys. 44: 244-5, 1953: Henry and Saini. Cereal Chem. 66: 362-365, 1989; Schnyder, New Phytol 123: 233-245, 1993). Wheat grain has been reported to contain 0.6-2.6% (w/w) fructan.
Fructan is synthesized directly from sucrose as the sole precursor, without any known involvement of phosphorylated sugars or nucleotide co-factors, by the activity of specific fructosyltransferases (FTs). Synthesis generally occurs in vacuoles, outside of the plastid, and accumulation of fructan occurs in vacuoles of both photosynthetic and storage cells. Fructan synthesis in plants is initiated by a sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) using sucrose both as fructosyl donor and acceptor to produce 1-kestose, the shortest β(1-2) linked fructan) and glucose. 1-SST is found in all fructan-producing plants. Longer chain inulins are formed by the action of a second enzyme, fructan:fructan 6-fructosyltransferase (1-FFT, EC 2.4.1.100) which adds fructosyl residues by β(1-2) linkages. 1-FFT can use 1-kestose or fructans as fructose donors and therefore can transfer fructosyl residues from one fructan chain to another. Synthesis of the neo-series fructans requires fructan:fructan 6G-fructosyltransferases (6G-FFT). In the case of cereals such as wheat and barley, the next step of fructan synthesis is mediated by a sucrose:fructan 6-fructosyltransferase (6-SFT, EC 2.4.1.10) which transfers a fructosyl unit from a further sucrose molecule to fructan with a β(2-6) linkages, to extend the fructan polymer. Fructosyl transfer to 1-kestose, the smallest branched fructan, forms the tetrasaccharide bifurcose. It remains to be shown whether or not additional FTs are involved in fructan synthesis of grasses or cereals, but the combined action of 1-SST, 1-FFT, 6-FFT and 6G-FFT may be involved in graminan synthesis.
Many plant fructosyltransferases have been sequenced during the last few years, and the data clearly indicate a high homology to the vacuolar, acid invertases (β-fructosidases). These enzymes are all members of the glycoside hydrolase family 32 (GH32) and share three highly conserved regions characterized by the motifs (N/S)DPNG (also called β-fructosidase motif), RDP, and EC. The aspartate of the (N/S)DPNG motif provides a nucleophile in the catalysis, the glutamate of the EC-motif as a proton donor, and the aspartate of the RDP motif as transition state stabilizer in the transfructosylation reaction.
Fructans are catabolised by fructan exohydrolases (FEH; EC 3.2.1.80) which are specialized for fructans, and invertases such as acid invertase (EC 3.2.1.26) which hydrolyse sucrose. Genes encoding fructan exohydrolase have been isolated from wheat (Van den Ende et al. Plant Physiol. 131(2): 621-631, 2003). Other enzymes such as sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) are associated with fructan remobilization from the stems.
Fructans are non-starch carbohydrates with potentially beneficial effects as a food ingredient on human health (Tungland and Meyer, Comprehensive Reviews in Food Science and Food Safety, 2: 73-77, 2002; Ritsema and Smeekens, Curr. Opin. Plant Biol. 6: 223-230, 2003). The human digestive enzymes α-glucosidase, maltase, isomaltase and sucrase are not able to hydrolyse fructans because of the β-configuration of the fructan linkages. Furthermore, humans and other mammals lack the fructan exohydrolase enzymes that break down fructans and therefore dietary fructans avoid digestion in the small intestine and reach the large intestine intact. However, bacteria there are able to ferment fructans and thereby utilize them as, for example, an energy or carbon source for growth and production of short-chain fatty acids (SCFA). Dietary fructans therefore are able to stimulate the growth of beneficial bacteria such as bifidobacteria in the colon, which aids in prevention of bowel disorders such as constipation and infection by pathogenic gut bacteria. Dietary fructan also enhances nutrient absorption from diets, particularly calcium and iron, possibly via production of SCFA which in turn reduce luminal pH and modify calcium speciation and hence solubility, or exert a direct effect on the mucosal transport pathway, thereby improving the mineralization of bone and reducing the risk of iron deficiency anaemia. In addition, a high-fructan diet can improve the health of patients with diabetes and reduce the risk of colonic cancers by suppressing aberrant crypt foci which are precursors of colon cancer (Kaur and Gupta, J. Biosci. 27: 703-714, 2002).
Attempts have been made to enhance fructan production in transgenic plants by introduction and expression of genes encoding 1-SST and 1-FFT. Generally, fructan accumulation levels were less than 2% (w/w) for plants transformed with bacterial genes and less than 1% (w/w) using plant genes. In some exceptions, concentrations of 6-16% on a fresh weight basis were achieved and compare favourably with naturally occurring maximal starch and fructan content in leaves and tubers (Sevenier et al., Nature Biotechnol. 16: 843-846, 1998; Hellwege et al., Proc. Natl. Acad. Sci. U.S.A. 97: 8699-8704, 2000). Transformants expressing bacterial fructan synthesis genes sometimes exhibited aberrant phenotypes such as stunting, leaf bleaching, necrosis, reduced tuber number and mass, tuber cortex discoloration, reduction in starch accumulation, and chloroplast agglutination.
There is therefore a need for efficient production of fructan from plant sources at low cost.